Investigation and application of liquid chromatography mass spectrometry in the analysis of polar, less volatile and thermal unstable organic pollutants in environmental and biological samples 2

2. Chapter Two
Experimental Section

This chapter describes the instrumentation, chemicals and procedures used throughout
this work, unless specified otherwise in a particular chapter.

2.1 INSTRUMENTATION

2.1.1 Sample Preparation Techniques: MAE and SFE

Microwave-assisted extraction was carried out using a MarsX (CEM, Matthews, NC,
USA, 1200-Watt) laboratory microwave extraction system equipped with a solvent
detector. The instrument is able to extract concurrently fourteen solid samples in PTFE
extraction vessels under identical extraction conditions. It controls either pressure or
temperature depending on which parameter reaches its control set point first.

SFE in the dynamic mode was performed using a Jasco (Tokyo, Japan) PU-980 HPLC
pump. A Trace ThermoQuest (Rodano, Italy) Series 2000 GC oven was used to produce
the required critical temperature of CO
2
. A 10-ml stainless steel sample cell (Jasco) was
installed in the GC oven. Methanol was added to the CO
2
at intervals with a second PU-
980 HPLC pump.




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2.1.2 Sample Measurement Systems: HPLC-UV, LC-MS and GC-ECD

The HPLC system consisted of a Shimadzu (Kyoto, Japan) LC-6A pump, a Rheodyne
(Cotati, CA, USA) 7010 injector equipped with a 20-µl loop, a Shimadzu SPD-6AV UV-
VIS detector and a Shimadzu C-R6A integrator.

The LC-API-MS analyses were performed with a Thermo Separation gradient HPLC
system (Model SCM 1000) coupled to a Finnigan MAT LCQ ion-trap mass spectrometer
(all ThermoQuest, San Jose, CA, USA). The instrument was initially tuned based on a
mixture of caffeine, L-methionyl-arginyl-phenylananyl-alanine acetate·H
2
O and a
mixture of perfluoroalkoxycyclotriphosphazenes in both positive and negative ionization
modes as suggested by the manufacturer.

GC analysis was performed by a Hewlett-Packard (Palo Alto, CA, USA) 5890 Series II
gas chromatograph equipped with a 63Ni electron-capture detector. Separations were
conducted using a DB-5, 30 m × 0.32 mI.D. capillary column (0.25 µm stationary phase
thickness) (J&W, Folsom, CA, USA). The carrier gas was purified nitrogen at a flow rate
1.5ml/min.





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2.2 REAGENTS

Analytical-grade PAHs (naphthalene and phenanthrene) were purchased from Supelco

(Bellefonte, PA, USA) or Ultra-Scientific (North Kingston, RI, USA). The stock standard
solutions were prepared in acetone at concentrations of 2.0 mg/ml for each compound
and stored at - 4°C. Working solutions were prepared by diluting the stock solutions with
acetone.

Polychlorinated biphenyls (PCB-1242 and PCB-1248) were purchased as individual
standard stock solutions containing a nominal concentration of 100 µg/ml in methanol
from Ultra-Scientific.

Atrazine (purity 98%) and simazine (purity 99%) were purchased from Supelco. Stock
solutions (1000 µg/ml) were prepared by dissolving the solid standards in acetone and
stored under refrigeration. Working solutions were obtained by diluting with acetone.

The carbamates: propoxur (purity 99%), methiocarb (purity 99%), propham (purity
99.5%), thiuram (purity 98%) and chlorpropham (purity 99.5%) were supplied by
ChemService (West Chester, PA, USA). The stock solutions containing each compound
at 1000 µg /ml were prepared in methanol and diluted with the same solvent to obtain
working solutions at various concentrations. They were stored at 4°C.


45
All solvents used in this study were either pesticide-grade or HPLC-grade and obtained
from Fischer Scientific (Fair Lawn, NJ, USA). The water used (thereafter referred to as
ultrapure water) was purified using a Milli-Q (Millipore, Bedford, MA, USA) water
purification system. All the samples (solutions and extracts) were filtered through 0.45-m
membranes (Millipore) and degassed in an ultrasonic bath before use.

2.3 PROCEDURES

2.3.1 Preparation of Water Samples

Natural water samples were collected from local sites. They were filtered through a 0.45-
µm membrane (Millipore) to remove particulate matter before use.

Freshly spiked water samples were prepared by adding an appropriate volume of spiking
solutions into the natural water samples prepared above and ultrapure water. All the
samples were degassed by an ultrasonic bath before LC-MS analysis.
2.3.2 Preparation of Soil Samples
2.3.2.1. Blank soils

Blank soils, collected from local sites were air-dried, pulverized and sieved through a 60-
mesh sieve. In order to remove possible traces of PAHs, PCBs, triazines, carbamates and
other organic contaminants, 100 g of each of the soil sample was immersed in 200 ml

46
each of methanol, acetone, dichloromethane and n-hexane for at least 24 hr sequentially.
The treated soil was spread out on a tray and air-dried for 8 hr in a fume hood to remove
as much solvent as possible. Finally, it was determined that there were no detectable
levels of the target analysts in soil samples before spiking.

2.3.2.2. Freshly spiked soils

Freshly spiked soil samples were prepared by adding an appropriate volume of spiking
solutions into the soils. To ensure that the analytes were well distributed, a reasonable
amount of acetone was added to moisten the soil and careful agitation was performed.
These standards were prepared 10-14 days prior to soil analysis.

2.3.2.3. Aged spiked soils

Aged spiked soil samples were obtained by storing the above spiked soil in bottles in a

dry, dark location for 60 days. It was assumed that the contaminants were uniformly
distributed in the sample and that, because the soil still retained residual moisture
throughout the storage period, any analyte-matrix interactions would have occurred, over
the weathering period, to a similar extent to those in real contaminated soil with similar
properties.




47
2.3.3 Preparation of Biological Samples

Goldfish, tortoises (Trachemys scripta elegans), and green alga (Sea lettuce: Ulva
lactuca) were purchased in a local market. In order to remove possible trace organic
contaminants prior to preparation, all of them were washed in fresh running water and
then rinsed with deionized water.

The fish samples were grinded in a mortar and pestle with liquid nitrogen until a
homogenous fine powder was obtained. In order to meet the fortification standards for
recovery and analytical precision, a portion of each fish sample was added an appropriate
volume of standard solution, thereby obtaining the required levels of target analytes. To
ensure that the analytes were well distributed, enough methanol was added to just
moisten the samples that were then stirred. After freeze-drying for 24 hours, the fine
powders were accurately weighed and placed in polyethylene flasks.

Tortoises were removed from their shells after being submerged in liquid nitrogen, and
tissues were then crushed and lyophilized. Spiked tortoise samples were prepared in a
similar manner as the fish. The remainders of the fish and tortoise samples were stored at
-20 °C for later analyses. All samples were analyzed as freeze-dried powders.

Sea lettuce was crushed until a homogeneous mass was obtained. Spiked samples used
for recovery determination were prepared via the addition of standard stock solutions to
the homogenized specimen (30 g) that was then left for 1 hour to allow the spiked

48
solution to penetrate the material. The samples were then immediately freeze-dried for
24h, and finally stored in polyethylene bottles after being weighed accurately. No sieving
was performed before analysis.

To assess possible contamination from sample preparation, together with each series of
samples, blanks were made to ensure there were no detectable levels of the target
analytes in each freeze-dried blank samples before spiking.

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